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The Reaction Sequence in Ergothioneine Biosynthesis: Hercynine As an Intermediate*

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The Reaction Sequence in Ergothioneine Biosynthesis: Hercynine As an Intermediate* THE JOURNAL OF BIO~~ICAL CHEMISTRY Vol. 237, No. 5, May 1962 Printed in U.S.A The Reaction Sequence in Ergothioneine Biosynthesis: Hercynine as an Intermediate* AMIR ASKARI AND DONALD B. MELVILLE From the Departments of Biochemistry, Cornell University Medical College, New York, New York, and the University of Vermont College of Medicine, Burlington, Vermont (Received for publication, January 9, 1962) Downloaded from Ergothioneine can be synthesized by the mold, Neurospora provide data which show that hercynine is an intermediate com- crassa, from the amino acids, histidine, methionine, and cysteine pound in the biosynthesis of ergothioneine. (1). The intact histidine molecule is used in the synthesis, and EXPERIMENTAL PROCEDURE the three methyl groups of the betaine moiety can be derived http://www.jbc.org/ from methionine by a triple transmethylation process (2). Cys- Materiak-L -Histidine-2 - Cl4 dihydrochloride was prepared teine appears to be an immediate precursor of the sulfur atom from NaC14N (4, 5). Sodium formate- was purchased from of the thiolimidazole ring. In this paper we present data which Tracerlab, Inc., Boston, Massachusetts. Ergothioneine was pur- establish the sequence of these biosynthetic reactions. chased from Nutritional Biochemicals Corporation, Cleveland, Of the several possible synthetic routes, the most straight- Ohio, and was recrystallized from aqueous ethanol. Methods-N. crassa (ATCC 10336, wild type A) was grown in forward involve as intermediates either thiolhistidine or hercy- at UNIVERSITY OF TOLEDO LIBRARIES on March 24, 2016 nine, the betaine of histidine, according to the depicted reaction a chemically defined medium (6) in shaken cultures as previously schemes (Scheme 1). described (1). Ergothioneine was extracted from the dried CH===C-CH2rCH-COOH CH=C-CH+H-COOH I k NH NH 2 Cyst&e N AH I&H* ’ NC/ I SH Histidine Thiolhistidine ethionine ethionine i? i? CH=C-CHe--CH-COO- CH=+CH+H-COO- Hercynine Ergothioneine SCHEME 1 The sequence of reactions involving thiolhistidine as an inter- mycelium with hot water and was purified by chromatography mediate has not been substantiated by studies which have been on alumina (7, 8). Column fractions were analyzed for ergo- done with thiolhistidine-2-P (1). The alternative pathway thioneine by the modified Hunter diazo reaction (9). Dried through hercynine has not been explored heretofore. Although aliquots in steel planchets were examined for radioactivity with hercynine was identified several years ago as a constituent of a mica window counter. mushrooms (3), the substance has not been well characterized, Preparation of Hercynine-Hercynine was prepared by the and no evidence for its biological relationship to ergothioneine oxidation of ergothioneine with ferric chloride (10). A solution has been adduced. In this paper we describe the preparation of of 100 mg of ergothioneine in 0.5 ml of water was mixed with 672 crystalline hercynine, including the CWlabeled compound, and mg of FeC13.6Hz0 dissolved in 6 ml of water. The mixture was heated under reflux for 30 minutes and then was cooled to room * This work was aided by grants from the National Science temperature. An aqueous 10% solution of Na2C03 was added, Foundation and the National Institute of Allergy and Infectious a few drops at a time with centrifugation, until the supernatant Diseases, United States Public Health Service. Requests for reprints should be addressed to Dr. Donald B. Melville, Depart- solution showed no further precipitation on the addition of ment of Biochemistry, University of Vermont College of Medicine, Na2C03. The final supernatant solution was removed, and the Burlington, Vermont. precipitate was washed twice with 3-ml portions of water. The 1615 1616 The Reaction Sequence in Ergothioneine Biosynthesis Vol. 237, No. 5 combined solutions were evaporated in a vacuum to dryness. paper chromatography with n-butanol-methanol-benzene-water The residue was extracted with three 4-ml portions of hot metha- (1:2:1:1) as the solvent. nol, and the combined extracts were filtered, made acid to lit- A 75-pg sample of the product, prepared as a thin film for mus with a few drops of concentrated HCl, refiltered, and evapo- measurement of radioactivity, showed 2180 c.p.m. A 150.pg rated to dryness in a vacuum. The residue was extracted with sample was added to an aqueous solution of 5 mg of nonisotopic three 2-ml portions of dimethylformamide, and the extracts were ergothioneine and 5 mg of ISa$04, and the mixture was treated combined, filtered, and evaporated to dryness in a vacuum. with an excess of bromine-water. The addition of barium chlo- This residue was chromatographed on a column of 20 g of alu- ride yielded barium sulfate, which was collected as a pad on filter mina with 100 ml of 80% ethanol as the solvent, 5-ml fractions paper and showed 9 c.p.m. being collected. Aliquots of each were tested with the modified Biosynthesis of Ergothioneine from Doubly Labeled Hercynine- Hunter diazo test for ergothioneine. Those fractions giving a Cl-A 48-hour culture of N. crassa was washed with sterile yellow color in the test were combined, the solvent was removed water and then was suspended in a sterile solution of 6 mg of the under reduced pressure, and the residue was extracted with three hercynine-Cl4 in 100 ml of water. The mixture was shaken for l-ml portions of methanol. Ether was added to the combined 24 hours, and the mycelium was collected, washed with water, Downloaded from filtered extracts until the solution was turbid. On standing and autoclaved with 35 ml of water containing 10 mg of non- overnight in the refrigerator, the solution deposited rosettes of isotopic ergothioneine. The aqueous extract was chromato- small rod-shaped crystals which were washed with ether and graphed on alumina with 75% ethanol-l% formic acid, and the dried. The crystals weighed 35 mg; an additional 10 mg were ergothioneine-containing fractions were combined and chromato- obtained from the mother liquors. For analysis the material graphed twice on alumina, each time with 80% ethanol as the was recrystallized twice from methanol-ether mixtures. A test solvent. Analysis of aliquots from the effluent fractions from http://www.jbc.org/ for ionic halogen was negative. the last column showed excellent correspondence between ergo- GH1502N3 (197.2) thioneine and radioactivity determinations. These fractions were combined, 20 mg of nonisotopic ergothioneine were added, Calculated: C 54.82, H 7.67, N 21.31 and the ergothioneine was crystallized from aqueous ethanol to Found : C 54.72, H 7.85, N 21.04 yield 23.6 mg of radioactive product. Recrystallization did at UNIVERSITY OF TOLEDO LIBRARIES on March 24, 2016 Hercynine prepared in this fashion is a colorless substance which not decrease the specific activity. The thrice crystallized com- is readily soluble in water, methanol, hot ethanol, acetone, and pound was used for degradation to thiolurocanic acid and tri- dimethylformamide, but is insoluble in ether, benzene, chloro- methylamine by treatment with hot concentrated NaOH as form, and ethyl acetate. It is hygroscopic, particularly when described previously (1). From 8.8 mg of the ergothioneine impure. The compound melts with decomposition at 235-237” there were obtained, after crystallization of the reaction products (corrected) on the micro melting point stage, with charring to constant specific radioactivities, 1.6 mg of thiolurocanic acid above 210”. No absorption occurs in the ultraviolet range at and 1.0 mg of trimethylamine chloroplatinate showing 302 c.p.m. wave lengths longer than 230 mp. [a]: +51.5” (c = 1.49 in and 116 c.p.m., respectively. water). In order to compare these radioactivities with that of the Preparation of Doubly Labeled Hercynine-Cl-Hercynine doubly labeled hercynine, 150 pg of the W-labeled hercynine labeled with Cl4 in both the imidazole ring and the methyl groups were mixed with 12.6 mg of nonisotopic hercynine, and the mix- was obtained by the degradation of doubly labeled ergothio- ture was subjected to treatment with hot concentrated alkali as neine-Cl4, which in turn was prepared biosynthetically by grow- described for ergothioneine. Trimethylamine was readily lib- ing N. crassa in the presence of formate-C14. To each of 10 erated under these conditions and was purified to constant flasks, each containing 100 ml of growth medium, were added radioactivity as the chloroplatinate. For the isolation of uro- 5.44 mg (0.08 mc) of sodium formate-CX4. To one of the flasks canic acid, the alkaline reaction mixture was acidified, evaporated approximately 0.25 me of carrier-free sulfuric acid-S35 was also to dryness in a vacuum, and extracted with dimethylformamide. added. N. crassa was grown in the media from spore inocula Urocanic acid was precipitated from the filtered extract by the for 96 hours. Each mycelium was then shaken for 24 hours in addition of ether and was purified to constant radioactivity by 100 ml of a sterilized aqueous solution of isotopic formate, or repeated crystallization from dimethylformamide-ether mixtures. formate and sulfate, in the same concentrations as were used in The final product was obtained as 1.8 mg of fine needles, micro the growth medium. Each mycelium was then collected on a m.p. 222-226”, with a radioactivity of 276 c.p.m. The trimeth- filter and heated in an autoclave at 15 pounds of pressure for 30 ylamine chloroplatinate weighed 4.8 mg and showed 362 c.p.m. minutes with 30 ml of water. The mycelia were washed twice The radioactivities of the doubly labeled hercynine-Cl4 and with small portions of hot water, and the combined extracts and the ergothioneineCi4 synthesized from it by N. crassa are com- washings were evaporated to dryness in a vacuum after the addi- pared in Table I.
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